Neisseria gonorrhea infection is one of the most prevalent sexually-transmitted bacterial diseases reported in humans. In response to this major health problem, numerous methods for detection of Neisseria gonorrhoea have been developed.
Most currently available procedures for the determination of gonococcal infection rely primarily upon culture techniques. Typical culture techniques include procedures described in Criteria and Techniques for the Diagnosis of Gonorrhea, published by the Center for Disease Control, Atlanta, Ga. In such culture techniques, a specimen, e.g., a urethral or cervical sample, is placed on an acceptable culture medium, e.g. Thayer-Martin medium. The cultures are incubated at 37.degree. C. in a 5% carbon dioxide atmosphere for 24 to 48 hours. The culture plates are then inspected for the appearance of Neisseria gonorrhoea colonies. Suspect colonies are gram-stained and tested for oxidase activity. Generally, presumptive diagnosis of gonococcal infection in males is determined by obtaining urethral cultures which exhibit oxidase-positive colonies of gram-negative "coffee bean" shaped diplococci when cultured on Thayer-Martin medium. In females, gonococcal infection may be diagnosed by examining cervical cultures on Thayer-Martin medium wherein oxidase-positive colonies of gram-negative diplococci appear. Organisms from presumptively identified colonies of Neisseria gonorrhoea are frequently confirmed by sugar fermentation, fluorescent antibody staining or coagglutination. However, such culture procedures are laborious, time consuming and are generally limited to detection of living cells. When culture methods are utilized, a specimen may be taken at one location and shipped to a laboratory, usually at another location, where the organisms are cultured and identified. Thus, these culture procedures may require several days before results are obtained. Furthermore, results obtained from culture procedures may be erroneous, if rather exacting conditions for preservation, shipment and culturing of the bacteria are not followed.
The genetic information of all organisms is stored in specialized molecules called DNA. The unique structure of DNA was first described by Watson and Crick in 1953. The DNA molecule consists of two linear polymeric strands, intertwined to form a double helix. Each strand is composed of alternating sugar and phosphate groups, stabilized by hydrogen bonding between pairs of nucleotide bases: Adenine (A), guanine (G), cytosine (C), and thymine (T). The two strands are complementary, with A always bonding to T on the opposite strand and G always bonding to C. Thus, if the sequence of bases in one strand is known, the sequence of its partner is also known. The sugar and phosphate groups maintain the structural integrity of the DNA molecule and remain constant throughout the molecule. Only the number of base pairs and the sequence in which the base pairs occur will vary. It is the precise number and sequence of bases along the strand which encodes all genetic information. Hence, the physical and biological nature of all organisms is determined by the unique sequence of nucleotide bases in its DNA. The identity of an organism can be determined, and even minor differences between related organisms can be detected if the sequence of bases responsible is known and can be observed.
The double-stranded DNA molecule is normally very stable; however, the two complementary strands can be separated (denatured). In vivo, natural processes such as DNA replication (synthesis of identical "daughter" molecules) and transcription (synthesis of messenger RNA) require that the two strands separate and serve as templates. In vitro, DNA can be denatured by treatment with heat or extremes of pH. These simple procedures break the hydrogen bonds which hold the complementary pairs together while preserving the integrity of the base sequences in the relatively stable single-stranded DNA. Under appropriate conditions, because of the nucleotide complementarity and hydrogen bonding, the single strands will rewind (reanneal). This process in which a double-stranded molecule is formed by specific sequence-dependent interaction of complementary single strands is called nucleic acid hybridization.
Nucleic acid probes have been used a research tool for detecting sequences of DNA. These probes exploit the ability of complementary strands of nucleic acid to hybridize and form one strand of nucleic acid. Nucleic acid probes are specific nucleic acid sequences that search for complementary sequences in a pool of single-stranded nucleic acid. Under proper conditions, the complementary strands collide, complementary sequences recognize each other and reform double-stranded molecules. The nucleic acid probe can be labelled using either a radioisotope or one of several nonradioactive labels, thereby allowing visualization of the nucleic acid hybridization reaction.
A recently developed method of detecting gonococcal infection employs a polynucleotide probe to detect nucleic acid from the gonococcal cryptic plasmid. (See DNA Hybridization Technique for the Detection of Neisseria gonorrhoea in men with urethritis. The Journal of Infectious Diseases, vol. 148 (3): 462-471, (1983). A drawback to this method is the fact that not all of the known strains of Neisseria gonorrhoea contain this plasmid. Whether a particular strain contains the plasmid is dependant to some extent on the geographical area in which the strain is found. Thus, this method can only detect infection with strains having the plasmid and will not detect strains that do not contain the plasmid. The test will not be reliably accurate if used in areas where strains do not have the plasmid and will not detect the presence of infection in areas where the plasmid is present in the bacterium, but individuals have brought the infection to the area from one where the strain of gonorrhea does not have plasmids. To avoid these drawbacks, it would be useful to have methods of detecting N. gonorrhoea which use chromosomal DNA which will be present in all strains of the bacteria, rather than plasmid DNA which is present in only some strains.
More recently, a chromosomal DNA test has been disclosed in European patent application number 87101215.9, filing date 29.01.87, date of publication 23.09.87, claiming U.S. priority date 30.01.86. Nucleic acid probes disclosed in the application contain fragments of chromosomal DNA from N. gonorrhoea. It is reported that these can be used in nucleic acid hybridization methods to detect the presence of N. gonorrhoea in biological specimens. The fragments of chromosomal DNA used in the probes were characterized as being specific for N. gonorrhoea by comparative hybridization with chromosomal DNA from N. meningitides. N. gonorrhoea DNA fragments of up to about 1,300 base pairs which had a low percentage of hybridization with N. meningitides DNA were considered to be specific for N. gonorrhoea and useful for detecting the bacterium in diagnostic methods. Although this method may be sensitive to N. gonorrhoea, the long length of the nucleic acid fragments in the probes increases the likelihood that the sequences may be complementary to DNA sequences of other species and thus hybridize with them, producing false positive results in methods utilizing the probes. A DNA fragment containing fewer than about 12 nucleotides is believed to have insufficient complexity to be specific for a given organism; however, a much longer fragment has an increased likelihood of containing within it subsequences which are specific for another organism. To overcome this drawback of the longer probes, nucleic acid probes of shorter length are needed to detect the presence of N. gonorrhoea in biological specimens. Also, there is no suggestion whatsoever in this European patent application of the particular nucleic acid sequence of these fragments. Thus, these restriction fragment probes must be isolated from the chromosomal DNA and cannot be chemically synthesized de novo.
There is also a need for sensitive tests which have the ability to detect either broad or narrow groups of related species. The genus Neisseria includes two species that are pathogenic for humans, N. gonorrhoea and N. meningitidis. Although N. gonorrhoea is isolated from many patients with asymptomatic infections, it is always considered to be a pathogen. On the other hand, N. meningitidis may be isolated from the throat or nasopharynx in a small proportion of healthy individuals as well as patients with meningococcal disease. Other Neisseria species such as N. sicca, N. lactamica rarely cause disease but may be part of the normal flora and therefore must not be confused with gonococcus or meningococcus.
Accordingly, it is an object of the invention to provide means for accurate, rapid detection of N. gonorrhoea in biological specimens. It is a further object of the invention to provide nucleic acid probes for use in methods of detecting N. gonorrhoea in biological specimens which overcome the problems associated with nucleic acid probes available to date.
The advantages of the methods and probes as described herein are manifold. Nucleic acid hybridization methods which use the nucleic acid probes of the invention can be done on solid supports which require small quantities of reagents, and thus should be less expensive to perform than tests requiring larger amounts of reagents. Further advantages of this technology include the ability to detect the target organism regardless of its metabolic state, the ability to detect either broad or narrow groups of related species, and the potential for developing a single rapid procedure that uses the same basic format for all tests.